The project includes structural studies of viruses, viral proteins, and receptors. During the period from July 1995 to June 1996, there were only three visits, due to the long down period. Studies were carried out on the following structures. (1) Polyomavirus. Polyoma is a DNA virus; its structure and that of the closely related simian virus 40 were determined in previous years using data collected at CHESS. Studies in this period included crystals of complexes of the virus with receptor fragments. The results have implications for the mechanism of viral entry and for viral pathogenesis. (2) Transferrin Receptor. This protein mediates uptake of iron into cells. Data were collected from crystals of recombinant, soluble receptor. (3) Sindbis virus. This is an "enveloped" virus, with a bilayer membrane. The crystals diffract only to low resolution, but there is an on-going effort to obtain better diffraction. (4) Reovirus cores. The reovirus core is a transcriptionally active particle that synthesizes and modifies mRNA. The large particle size makes this a "frontier" problem in virus crystallography. Summary of Progress (1) Polyomavirus. Diffraction measurements were carried out on complexes of two different oligosaccharide analogs with three different viral strains. Each was studied over a range of concentrations, in order to determine affinities (by measuring the concentration of half-maximal occupancy). The highly virulent LID strain, which differs at only two key positions in VP1 from the standard RA strain, binds the receptor more weakly, extending an evident negative correlation (indicated by earlier results) between affinity and pathogenicity (in murine hosts). It appears that rapidity of spread in the animal, rather than efficiency of binding and entry, is the critical determinant of pathogenicity. This result is likely to be true of other viruses, with important consequences for thinking about antiviral drug strategies. (2) Transferrin receptor. Cryo-crystallography has proved to be critically important for collecting accurate data from crystals of the ectodomain of human transferrin receptor, expressed as a secreted recombinant product in CHO cells. The crystals are in space group P212121, a = 365 _, b = 224 _. c = 105 _; there are eight 70 kD fragments in the asymmetric unit. The diffraction, using the F1 beamline and image-plate recording, extends to 3.2 _. Crystal damage, even at -160x C, limits data collection to about 10x per crystal, but diffraction data recorded from multiple crystals can be merged with good accuracy. A native data set has been assembled, and a derivative search is in progress. (3) Sindbis virus. Crystals of various strains, grown under a series of conditions, were examined. We were successful in determining conditions for cryogenic cooling, but even in the best cases, diffraction extended only to 30 _. Because of interest in understanding the relationship between the viral proteins and the lipid bilayer, we have undertaken to assemble a 30 _ resolution data set, to be used with starting phases from cryoEM. (4) Reovirus cores. The crystals are in space group F432, a = 1249 _. Data can be recorded to at least 7 _ at -160x C. Part of a data set have been assembled, and further data collection is planned during June 1996. The overall strategy is to use reconstructed images from cryoEM to obtain a phase start and one or more derivatives to aid in phase extension.